Evaluation of mineral status in hypothyroidism in a tertiary care centre

Background: Hypothyroidism is known to be the commonest form of endocrine disorders and has been linked with disturbances in various minerals metabolism. Calcium, phosphorus and magnesium and trace element zinc are required for many enzymes in various metabolic pathways which are directly or indirectly regulated by thyroid hormones. Aim and objectives of the study was to estimate serum zinc, calcium, magnesium and phosphorus in hypothyroid patients, with the objectives to evaluate any relationship with TSH and to compare them with euthyroid controls.Methods: The analytical cross-sectional study included 50 hypothyroid subjects with TSH levels >4.5 mcg IU/mL and 50 euthyroid subjects of 20-50 years in RMCH, Bareilly. TSH was estimated by ECLIA, serum calcium and phosphorus were estimated by autoanalyzer and serum zinc & magnesium by the kit method using semi autoanalyzer. All the biochemical parameters were expressed as median with Interquartile Range (IQR). Mann-Whitney test was applied to compare the parameters of cases and control. Spearman’s rank correlation coefficient 2-tailed was used to correlate the parameters among the cases.Results: A significantly decreased level of serum calcium and increased level of serum magnesium and phosphorus were observed in hypothyroid cases. A significant negative correlation between TSH and serum calcium while a significant positive correlation of serum magnesium and phosphorus with TSH was observed.Conclusions: The indexed study indicates the significant effect of overt or subclinical hypothyroidism over the mineral status of the body which may have inconsistent effect over the various metabolism and enzymes and thereby clinical manifestations.

Production and characterization of monoclonal antibodies to Newcastle Disease Virus.

Newcastle Disease (ND) is one of the major causes of economic loss in the poultry industry. Newcastle Disease Virus (NDV) is a single-stranded, negative-sense enveloped RNA virus (Fam. Paramyxoviridae; Order Mononegavirales). In the present study three monoclonal antibodies (MAbs) were produced by polyethyleneglycol (PEG)-mediated fusion of lymphocytes sensitized to NDV Bareilly strain and myeloma cells. NDV possesses ability to agglutinate erythrocytes of avian species. All the three MAbs designated as 2H7, 3E9 and 3G6 caused hemagglutination inhibition of NDV by specifically binding to NDV. The reactivity for all the 3 MAbs on indirect ELISA was found to be significantly higher than the antibody and antigen controls. On flowcytometry of HeLa cells infected with NDV using the MAbs as primary antibodies, there was a significant difference in the percentage of cells showing positive fluorescence compared to the mock control. One of the MAbs (3E9) was found to react with hemagglutinin-neuraminidase (HN) protein on western blot.

Comparative Study of Serum Lipid Profile in Normotensive Pregnant Women and in Patients with Pre-Eclampsia- A Case Control Study.

Background: The objective of the present study was to compare the changes in serum lipid profile in normal pregnant women with those in patients with pre-eclampsia. Methods: In this study total 140 study subjects were evaluated, 70 normotensive pregnant women as a control group and 70 pre-eclamptic patients as a study group. Study Subjects were between the age group of 20-35 years and in the second and third trimester of pregnancy. Fasting blood samples were collected and serum level of Triglycerides (TG), Total Cholesterol (TC), High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), and Very Low Density Lipoprotein (VLDL) were measured by enzymatic colorimetric method. Results: There was a significant rise in TC, TG, LDL and VLDL and significant decrease in HDL level in pre-eclamptic group as compared to normal pregnant women.Conclusion: The findings of the present study are consistent with previous studies suggesting an altered lipid profile has a potential role in the genesis of endothelial dysfunction and expression of pre-eclampsia.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Association of ABO blood group and breast cancer in Jodhpur.

There is a large amount of evidence that the ABO blood group system may play a role in disease etiology. However, in relation to breast cancer, these findings are inconsistent and contradictory. Present study was conducted for analysis to access ABO blood groups potential role of in breast carcinoma. The study was conducted on 206 clinically diagnosed breast cancer patients from Radiotherapy Department of Mathura Das Mathur Hospital in Jodhpur, from September 2006 to December 2007. The standard agglutination test was used to determine the blood groups. Association of ABO blood groups and risk of breast cancers was found out with Odd Ratios (ORs) with 95% Confidence Interval (CI). In reference of proportion of breast cancer in blood group AB [OR 1 with 95% CI 0.476 to 2.103), the breast carcinoma in blood group A [OR 7.444 with 95% CI 4.098 to 13.5222) was found at 7.4 times at higher risk than in blood group ‘AB’. Breast cancer was found minimum in blood group ‘AB’ and maximum in blood group ‘A’.

Development of dog mammary tumor xenograft in immunosuppressed Swiss albino mice.

Development and study of dog mammary tumour xenograft in immunosuppressed Swiss Albino Mice adds a new dimension in cancer research as dog tumors have many similarities with human tumors regarding progression, histopathology, molecular mechanism, immune response and therapy. Failure of the immune system to recognize and eliminate cancer cells leads to cancer progression and the fight between immune cells and cancer cells has a great role in understanding the mechanism of cancer progression and elimination. Rejection and acceptance of tumour xenograft depends on efficiency of CD4+, CD8+ and NK cell populations. In the present investigation, dog mammary tumor xenograft in cyclosporine-A and γ-irradiated, immunosuppressed Swiss Albino mice was developed and the immune cell status of graft accepted and rejected mice was assessed. It was observed that all the major immune cells (CD4+, CD8+ and NK cells) play an equal role in tumour rejection.

In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

The canine Parvovirus 2, non-structural 1(NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1(rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS–PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 × His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies.

In the present study recombinant VP3 (rVP3) was expressed in E.coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS–PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37ºC. The 6×His-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line.

Parvoviruses are small, 260-Å-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.